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p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy–lysosomal pathway and coordinate cell cycle and cell growth

A Publisher Correction to this article was published on 27 July 2021

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Abstract

Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27−/− cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27−/− cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.

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Fig. 1: Autophagy flux is promoted by p27 during prolonged amino acid starvation.
Fig. 2: p27 localizes on lysosomes following amino acid starvation.
Fig. 3: Lysosomal function decreases with p27 loss.
Fig. 4: p27 binds to LAMTOR1 and inhibits Ragulator assembly.
Fig. 5: p27 interferes with Ragulator functions.
Fig. 6: p27 participates in the inhibition of mTOR signalling in amino acid-deprived cells.
Fig. 7: Autophagy and mTOR are controlled by p27 in a LAMTOR1-dependent manner.
Fig. 8: Elevated mTOR activity in p27−/− cells confers resistance to amino acid starvation-induced apoptosis.

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Data availability

The datasets generated during and/or analysed in the current study are available from the corresponding author on reasonable request. Source data for all graphs and immunoblots of all figures are provided with the paper as Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank all of the members of the Besson and Manenti laboratories for stimulating discussions. We thank D. M. Sabatini (Whitehead Institute for Biomedical Research, Cambridge, USA), J. Debnath (UCSF, San Francisco, USA) and N. Mizushima (Metropolitan Institute of Medical Science, Tokyo, Japan) for providing reagents. We thank V. W. Rebecca (University of Pennsylvania, Philadelphia, USA) for technical advice regarding the PLA experiments. A.N. was supported by studentships from the Ministère de l’Enseignement Supérieur et de la Recherche and Fondation ARC pour la Recherche sur le Cancer. J.C. was supported by a studentship from the Region Midi-Pyrénées and Université Paul Sabatier—Toulouse III. R.T.P. was supported by a studentship from the Ligue Nationale Contre le Cancer. S.M.’s team is an ‘Equipe labellisée Ligue Nationale contre le Cancer 2016’. This project was supported by funds from the Ligue Nationale Contre le Cancer and Fondation ARC pour la Recherche sur le Cancer to A.B. A.B. is supported by an ‘FRM Equipes’ grant (grant no. DEQ20170336707) from the Fondation pour la Recherche Médicale.

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Authors

Contributions

A.N., P.J., C.C., J.C., R.T.P. and A.B. performed the experiments. A.N., C.J., P.C., S.M. and A.B. designed the experiments. A.N. and A.B. analysed the data and wrote the paper with contribution from all authors.

Corresponding author

Correspondence to Arnaud Besson.

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Extended data

Extended Data Fig. 1 Cytoplasmic p27 promotes autophagy in a CDK-independent manner in amino acid-deprived cells.

a, Immunoblots for p-S757 ULK1, ULK1 and Vinculin (loading control) in p27+/+ and p27−/− MEFs aa starved for the indicated times. The same extracts were probed for p-T172 AMPK and AMPK. b, p-T198 p27, p27 and Grb2 (loading control) immunoblots in MEFs starved for the indicated times. c, LC3B and β-actin (loading control) immunoblots in MEFs in full medium (0 h) or aa-starved for 1 h and 4 h ± 50 µM CQ for 1 h and 2 h, respectively. d, Quantification of autophagy flux (ratio of (LC3B-II + CQ/β-actin)/(LC3B-II-CQ/β-actin)) from n = 4 (0 h), n = 3 (1 h) or n = 6 (4 h) independent experiments as in c. e, p62 and Vinculin (loading control) immunoblots in MEFs in full medium (0 h) or aa starved for 48 h. f, Mean p62 fold change normalized to 0 h from n = 7 independent experiments as in e. g, Immunoblots for LC3B, p27 and β-actin (loading control) in MEFs retrovirally infected with mCherry-eGFP-LC3B ± 50 µM CQ for 2 h. h, LC3B and β-actin (loading control) immunoblot in p27+/+, p27−/−, p27CK- and p27S10A MEFs in full medium (0 h) or aa starved for 48 h ± 50 µM CQ for 2 h. i, Quantification of autophagy flux (as in d) in MEFs aa starved for 48 h from n = 11 (p27+/+ and p27−/−), n = 4 (p27CK-), and n = 3 (p27S10A) independent experiments as in h. p27+/+ and p27−/− data already appears in Fig. 1f. j, Immunoblots for LC3B, p27 and Grb2 (loading control) in p27−/− cells infected with empty vector or p27CK-. k, Quantification of autophagy flux (as in d) in cells aa deprived for 48 h. Graph shows mean from n = 2 independent experiments as in j, normalized to p27−/−+vector condition. a, b, c, e, g, h, j, Data presented are representative from n = 2 (a, j), n = 3 (b, c, g, h), n = 7 (e) independent experiments. d, f, i, Graphed data show means ± s.e.m. Statistical significance was evaluated by 2-way ANOVA followed by Bonferroni multiple comparison test (d), unpaired two-tailed Student’s t-test with Welch’s correction (f) or one-way ANOVA followed by Bonferroni’s multiple comparison test (i). Statistical source data and unprocessed blots are provided in Source data.

Source data

Extended Data Fig. 2 p27 localizes to lysosomes following amino acid starvation.

a, Quantification of p27 colocalization with LAMP2 (left) and LAMTOR1 (right), as described in Fig. 2a. Graphs show Manders’ overlap coefficient in cells in full medium (0 h) or aa-starved for 24 h from n = 69 (0 h) and n = 72 (24) h cells for p27/LAMP2, and n = 45 (0 h) and n = 48 (24 h) cells for p27/LAMTOR1 from 2 independent experiments. Statistical significance was evaluated by Mann-Whitney two-tailed test. b, PLA labelling of cells aa starved for 18 h with all the reagents except that only one primary antibody was added (single antibody controls) or in absence of primary antibodies (Probes only). F-actin was stained with phalloïdin. Representative images from n = 3 independent experiments. c, p27+/+ MEFs were transfected with LAMP1 siRNA for 24 h and then aa starved for 24 h before PLA staining. siRNA efficiency was evaluated by immunoblot for LAMP1 and β-actin (loading control). Graph shows mean ± s.e.m. number of PLA puncta per cell from n = 24 (Control) and n = 15 (siLAMP1) images from 3 independent experiments. d, p27+/+ MEFs were transfected with LAMTOR1 siRNA and treated as in c. siRNA efficiency was evaluated by immunoblot for LAMTOR1 and β-actin (loading control). Graph shows mean ± s.e.m. number of PLA puncta per cell from n = 24 (Control) and n = 19 (siLAMTOR1) images from 3 independent experiments. b-d, Scale bars are 50 µm. c, d, Statistical analyses were performed by unpaired two-tailed Student’s t-test with Welch’s correction. Statistical source data and unprocessed blots are provided in Source data.

Source data

Extended Data Fig. 3 p27 promotes autophagosome maturation but does not affect autophagogome–lysosome fusion or endocytosis.

a, LC3B and p62 immunostaining in p27+/+ and p27−/− MEFs aa starved for 48 h ± 50 µM CQ for 2 h. Dotted lines delineate LC3B-positive ring-like structures. b, Quantification of LC3B-positive ring-like aggregates per cell in MEFs aa deprived for 48 h as in a from n = 89 p27+/+ and n = 56 p27−/− cells examined from n = 3 in independent experiments. c, Quantification of LC3B dots colocalizing with p62 signal in cells as described in a from n = 4 (aa starvation) or 3 (aa starvation + CQ) independent experiments. d, LC3B and LAMP2 immunostaining in p27+/+ and p27−/− MEFs aa-starved for 48 h + 50 µM CQ for 2 h. Graphs display the fluorescence intensity (arbitrary unit) in each channel over the distance depicted by the arrows. e, Quantification of LAMP2 signal overlapping with LC3B dots in cells as described in d from n = 3 independent experiments. f, Images of DQ-BSA and Dextran-TRITC in p27+/+ and p27−/− MEFs in full medium. g, LAMP2 immunostaining in cells in full medium (0 h) or aa-starved for 48 h. h, Quantification of LAMP2 fluorescence intensity in cells as described in g from n = 208 (p27+/+ 0 h), n = 155 (p27+/+ 48 h), n = 131 (p27−/− 0 h) and n = 103 (p27−/− 48 h) from 3 independent experiments. Values were normalized to p27+/+ cells in the same condition. a, d, f, g, images presented are representative from n = 3 independent experiments. Scale bars are 10 µm. b, c, e, h, Graphs show means ± s.e.m. Statistical significance was evaluated by unpaired two-tailed Student’s t-test with Welch’s corrections (b, e) or by two-way ANOVA followed by Bonferroni’s multiple comparison test (c, h). Statistical source data are provided in Source data.

Source data

Extended Data Fig. 4 p27 binds to Ragulator on lysosomes.

a, Endogenous LAMTOR1 was immunoprecipitated from U251N cell lysates transfected or not with LAMTOR1 siRNA for 48 h. Rabbit IgG were used for control immunoprecipitations. Membranes for immunoprecipitates and extracts were probed for p27 and LAMTOR1. β-actin was used as loading control. Representative experiment from n = 4 independent experiments. b, Confocal images of colocalization of p27 with LAMTOR4 in digitonin-permeabilized MEFs in full medium (0 h) or aa starved for 24 h. p27−/− MEFs were used to set exposure time in the p27 channel. Graphs show the fluorescence intensity (arbitrary unit) of each channel over the distance depicted by the arrows. Representative images from n = 3 independent experiments. c, p27+/+ MEFs in full medium or aa-deprived for 18 h were subjected to PLA using anti p27 and LAMTOR4 antibodies and counterstained for LAMP2. PLA probed without primary antibodies were used as negative controls. Representative images from n = 2 independent experiments. d, p27+/+ and p27−/− MEFs in full medium or aa starved for 24 h were subjected to PLA using anti p27 and LAMTOR4 and counterstained for LAMP2. Representative images from n = 2 independent experiments. c,d, Corresponding single antibody PLA controls and probe only controls are shown in Extended Data Fig. 2b. b–d, Scale bars are 10 µm. Unprocessed blots are provided in Source data.

Source data

Extended Data Fig. 5 p27 controls RagA and mTOR recruitment to lysosomes.

a, RagA and LAMP2 immunostaining of p27+/+ and p27−/− MEFs in full medium or aa starved for 48 h. Graphs display the fluorescence intensity (arbitrary unit) in each channel over the distance depicted by the arrows. Representative images from n = 3 independent experiments. b, Quantification of the fluorescence intensity of RagA overlapping with LAMP2 signal normalized to p27+/+. Mean fluorescence intensity was analysed in n = 23 (p27+/+) and n = 22 (p27−/−) ROI (region of interest) from 3 independent experiments. c, Quantification of the fluorescence intensity of mTOR overlapping with LAMP2 signal as described in d, normalized to p27+/+ aa-starved for 48 h. Mean fluorescence intensity was analysed in n = 85 (p27+/+ -aa 48 h), n = 105 (p27+/+ -aa 48 h + aa 10 min), n = 81 (p27−/− -aa 48 h) and n = 93 (p27−/− -aa 48 h + aa 10 min) ROI from 2 (-aa 48 h) or 1 (-aa 48 h + aa 10 min) independent experiments. d, mTOR and LAMP2 immunostaining in p27+/+ and p27−/− MEFs either in full medium (0 h), aa starved for 48 h or aa starved for 48 h and re-fed with aa for 10 min prior to fixation. Representative images from n = 2 (-aa 48 h) and n = 1 (-aa 48 h + aa 10 min) independent experiments. b, c, Data are presented as means ± s.e.m. Statistical differences were evaluated by unpaired two-tailed Student’s t-test with Welch’s correction (b) or 2-way ANOVA followed by Bonferroni’s multiple comparison test (c). a, d, scale bars are 10 µm. Statistical source data are provided in Source data.

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Extended Data Fig. 6 Elevated mTOR activity in p27−/− cells confers resistance to amino acid starvation-induced apoptosis.

a, Immunoblots for cleaved caspase 3 and β-actin (loading control) in p27+/+ and p27−/− MEFs aa-starved for 48 h. Representative experiment from n = 3 independent experiments. b, Representative Incucyte images from 3 independent experiments of p27+/+ and p27−/− MEFs in full medium or aa-starved for 48 h ± 20 µM ZVAD caspase inhibitor. Scale bars are 200 µm. c, Quantification of apoptosis from n = 3 independent experiments as described in b, normalized to condition without ZVAD. Graph shows means ± s.e.m. Statistical significance was evaluated by 2-way ANOVA with Bonferroni’s multiple comparison test. d, Immunoblots for p27 and β-actin (loading control) of p27−/− cells infected with empty vector, p27 or p27CK-. Immunoblots representative from n = 4 (p27) and n = 5 (p27CK-) independent experiments. e, Quantification of apoptosis in p27−/− MEFs infected with empty vector, p27 or p27CK- as described in d aa starved for 48 h from n = 6 (Vector), 4 (p27) or n = 5 (p27CK-) independent experiments. Graph shows means ± s.e.m. Statistical significance was evaluated by Student’s t-test with Welch’s correction. f–h, Incucyte images (phase contrast and caspase-3/7 cleavage [green]) of (f) ATG5-inducible MEFs ± 10 ng/ml doxycycline in full medium or glucose or aa-starved for 48 h, (g) p27+/+ and p27−/− MEFs in full medium or aa-starved for 48 h ± 200 nM Torin1, or (h) p27+/+ and p27−/− MEFs transfected with control or LAMTOR1 siRNA in full medium or aa-starved for 24 h, illustrating the graphs in Fig. 8f, h, j, respectively. Images are representative from n = 6 (f), n = 8 (h) and n = 3 (h) independent experiments, respectively. a, f, g, h, Scale bars are 200 µm. Statistical source data and unprocessed blots are provided in Source data.

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Nowosad, A., Jeannot, P., Callot, C. et al. p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy–lysosomal pathway and coordinate cell cycle and cell growth. Nat Cell Biol 22, 1076–1090 (2020). https://doi.org/10.1038/s41556-020-0554-4

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